This is a re-submission of a competitive renewal application for the continuation of a more than twenty year old project to study DNA alkylation repair in eukaryotic cells. We propose to explore how one particular DNA repair pathway, namely base excision repair (BER) initiated by the alkyladenine DNA glycosylase repair enzyme (Aag in mice, AAG in humans; a.k.a. Mpg/MPG and Apng/APNG), contributes to the response of different cell types upon exposure to DNA damaging agents. Alkylating agents are cytotoxic, mutagenic, clastogenic, teratogenic and carcinogenic, and they represent one of the most potent and abundant classes of chemical DNA damaging agents in our environment. In addition, alkylating agents are present inside cells as normal cellular metabolites, and they are produced as byproducts of oxidative stress. Moreover, some of the most toxic alkylating agents are commonly used for the chemotherapeutic treatment of cancer patients. In the past funding period one of the most important things we learned (in addition to identifying a number of new pathways involved in protecting cells against alkylating agents) is that in certain cell types, expression of the Aag DNA repair enzyme, even at wild type levels, has the counter-intuitive consequence of rendering those cells extremely sensitive to the cytotoxic effects of DNA alkylation damage. In the next funding period we will seek to understand more fully what features of these specific cell types render Aag-initiated DNA repair so detrimental to cell survival. This will be achieved by pursuing the following four Specific Aims: Aims 1, 2 and 3 will exploit recent advances in the stem cell/regenerative medicine field for the in vitro differentiatin of mouse embryonic stem (ES) cells into specifically differentiated hematopoietic myeloid cells, retinal photoreceptor cells, and cerebellar granule cells, all of which display Aag-dependent alkylation sensitivity in vivo, unlike their ES cell progenitor that displays Aag-dependent alkylation resistance. In addition, as part of Aims 1 and 3, we will directly isolate differentiatig and differentiated cells from mice. All cell types will be tested for their sensitivity to two diffrent alkylating agents, and one non-alkylating agent, ultraviolet light. By isolating and characterizing (Aim 4) these isogenic cells whose phenotype has transitioned from Aag-dependent alkylation resistance (ES cells) to Aag-dependent alkylation sensitivity (myeloid, photoreceptor and granule neuron cells) we hope to learn how to better predict how cells and people will respond to alkylating agents ubiquitously present in the environment, and commonly used in the clinic. The health relatedness of the proposed project is that the information gained will contribute to furthering our goals to prevent cancer and other DNA damage-related diseases, while also contributing to improving how those diseases, especially cancer, are treated in the clinic.